4.4. MYC Immunohistochemistry

Immunolabeling of FFPE sections of all specimens utilized for NGS was performed by the Department of Pathology clinical laboratory using 5μm sections with a recombinant anti-c-Myc antibody (Abcam ab32072, clone Y69, dilution 1:400; Cambridge, UK) using the Ventana benchmark Ultra platform, ultraView kit (Tucson, AZ, USA), and standard protocols. In human studies, high MYC lymphoma was used as a positive control, while primary antibody was removed in negative controls. Specimens were scored as having no (0), weak (1), moderate (2), or strong (3) immunoreactivity, and the percentage of tumor cells staining was recorded. An h-score was calculated by multiplying the staining intensity score by the percentage (by decile) of positively-labeled tumor cells (or non-neoplastic sebaceous gland). Low h-scores were designated 0–99, and an h score of 100–300 was designated as high. Immunohistochemical scoring was performed independently by two pathologists (CP and CGE).

Eyelids from normal adult cadaveric C57B6 and CD1 mice (Charles River, Wilmington, MA, USA) were dissected and fixed in 10% NBF for 72 h at room temperature. Samples were processed, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E) by the JHMI Reference Histology Laboratory using standard protocols. Immunohistochemical analysis was performed on tissue sections using MYC (rabbit monoclonal, clone EP121, Epitomics, 1:600; St. Louis, MO, USA) as previously described [72]. In the murine studies, normal prostatic glandular epithelium and prostatic adenocarcinoma were used as negative and positive controls.